ARTICLE IN PRESS Deep-Sea Research I 57 (2010) 143–150 Contents lists available at ScienceDirect Deep-Sea Research I journal homepage: www.elsevier.com/locate/dsri High diversity of frenulates (Polychaeta: Siboglinidae) in the Gulf of Cadiz mud volcanoes: A DNA taxonomy analysis A. Hila´rio a,Ã, S.B. Johnson b, M.R. Cunha a, R.C. Vrijenhoek b a CESAM, Departamento de Biologia, Campus Univesita´rio de Santiago, 3810-193 Aveiro, Portugal b Monterey Bay Aquarium Research Institute, Moss Landing, California 95039-9644, USA article info abstract Article history: Frenulates are the most poorly known members of the family Siboglinidae (Polychaeta: Canalipalpata). Received 19 March 2009 These thread-like worms occur in reducing marine sediments worldwide, but they are often overlooked Received in revised form in benthic samples or too poorly preserved for adequate taxonomic evaluations. We report on a 29 September 2009 remarkable diversity of frenulates that were recently sampled from 13 mud volcanoes (350–3902 m Accepted 2 October 2009 deep) in the Gulf of Cadiz, off southern Iberia. Sampled with benthic coring devices, the bodies of these Available online 12 October 2009 long tubiculous worms were often broken or incomplete, making them difficult to identify Keywords: morphologically. Consequently, we employed DNA taxonomic methods to assess their diversity. Frenulata Mitochondrial cytochrome-c-oxidase subunit 1 (COI) sequences distinguished 15 evolutionary lineages Mud volcanoes inhabiting the Gulf of Cadiz. Only four of the lineages could be assigned to currently recognized Atlantic Diversity species; the remaining 11 may be new to science. This remarkable diversity of frenulates in a small DNA taxonomy geographical region is unprecedented and is hypothesized to result from environmental heterogeneity associated with the bathymetric and geochemical settings of these mud volcanoes. & 2009 Elsevier Ltd. All rights reserved. 1. Introduction et al., 2005). Vestimentiferans are relatively easy to collect with the aid of submersibles because they are large and often occur in The annelid family Siboglinidae (Polychaeta: Canalipalpata) clumps. Though Osedax are small, they occur on bones that are contains approximately 160 nominal species that inhabit deep- also relatively easy to sample with submersibles. Sclerolinum and sea reducing environments. Named after the Siboga Expedition the frenulates, on the other hand, are mostly small thread-like during which they were first collected (Caullery, 1914), these worms that are buried deeply in soft sediments. Their in situ tubeworms are among the dominant constituents of the inverte- observation is very difficult, so they are usually sampled as by- brate communities at hydrothermal vents, cold-water sulphide/ catch in benthic cores. Consequently, they are often poorly hydrocarbon seeps, and whale-falls worldwide (Braby et al., 2007; preserved or simply overlooked (Halanych, 2005). Southward et al., 2005; Tunnicliffe et al., 2003). Adult siboglinids The systematic position and nomenclature of Siboglinidae lack a digestive system and depend entirely on endosymbiotic Caullery (1944) was in a state of flux for many years (reviewed in bacteria for their nutrition (reviewed in Thornhill et al., 2008). Pleijel et al., 2009). Pogonophorans and vestimentiferans have at Most species have long and thin bodies that span oxic–anoxic various times been elevated to the rank of phylum, but current boundaries in the marine benthos, absorbing oxygen with an morphological, embryological and molecular evidence places anterior gill-like structure and reduced compounds (e.g., hydro- these worms firmly within the Polychaeta (Black et al., 1997; gen sulfide, methane or organic compounds) through their Halanych, 2005; Halanych et al., 2001; Kojima et al., 1993; posterior body (Goffredi et al., 2005; Southward et al., 2005). McHugh, 1997; Rouse, 2001; Rouse and Fauchald, 1997; South- Vestimentiferans, which dominate hydrothermal vents and cold ward, 1999; Young et al., 1996). As a result of these studies, most seep environments in the eastern Pacific and Gulf of Mexico, are researchers now recognize the family Siboglinidae as encompass- the most extensively studied siboglinids (Tunnicliffe et al., 2003). ing three discrete clades: the Frenulata; the Monilifera (=Vesti- Bone-eating worms of the genus Osedax, which were first reported mentifera+Sclerolinum as defined by Rouse (2001)); and Osedax in 2004, have been the subject of intense interest (Fujikura et al., (Rouse et al., 2004). Nonetheless, some researchers do not 2006; Glover et al., 2005b; Rouse et al., 2004, 2008; Southward recognize the new classification and continue to use the names Pogonophora and Vestimentifera to represent annelid classes (e.g., Bartolomaeus et al., 2005; Southward et al., 2005). Ã Corresponding author. Tel.: +351 234370969; fax: +351 234372586. The present study was stimulated by the discovery of E-mail address: [email protected] (A. Hila´rio). biologically diverse chemosynthetic communities in the Gulf of 0967-0637/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr.2009.10.004 ARTICLE IN PRESS 144 A. Hila´rio et al. / Deep-Sea Research I 57 (2010) 143–150 Cadiz, off the southern Iberian Peninsula (Cunha et al., 2001; solution of dNTPs, and sterile water to final volume. Amplifica- Rodrigues and Cunha, 2005). Ongoing explorations conducted by tions for all loci, which occurred with a Cetus 9600 DNA Thermal several research programmes (Akhmentzhanov et al., 2007; Cycler (Perkin-Elmer Corp., CT), used an initial denaturation of Weaver et al., 2004) have provided excellent opportunities to 95 1C/10 min, followed by 35 cycles of 94 1C/1 min, 55 1C/1 min, sample the fauna and extend our knowledge of the frenulates and 72 1C/1 min, and a final extension at 72 1C/7 min. PCR found at several mud volcanoes in this region. Based on their products were diluted in 40 ml sterile water and cleaned with unique morphological characteristics, a new frenulate genus, Multiscreen HTS PCR 96 Filter plates on a vacuum manifold Bobmarleya, and two new species, Bobmarleya gadensis and (Millipore Corp., Billerica, MA). PCR products were sequenced Spirobrachia tripeira, have already been described from the Gulf bidirectionally with the same primers used in PCR on an ABI 3100 of Cadiz (Hila´rio and Cunha, 2008). Morphological studies alone capillary sequencer using BigDye terminator v3.1 chemistry are not sufficient, however, to provide a precise accounting of (Applied Biosystems Inc., Foster, California). DNA sequences were frenulate species diversity in this region. Several putatively new proofread using Sequencher v 4.7 (Gene Codes Corp. Inc., Ann species can only be distinguished by subtle morphological Arbor, Michigan) and aligned and edited by eye with MacClade characters that are easily damaged during collection and dissec- v4.08 (Maddison and Maddison, 2005). tion. Furthermore, some diagnostic traits tend to vary with the age of individuals (Southward, 1969). Consequently, we employ 2.3. Phylogenetic analyses DNA taxonomic methods in the present study because they can be used to circumscribe and delineate distinct evolutionary lineages Published sequences (Table 2)fromOsedax rubiplumus, from incomplete or damaged individuals and from various life- Sclerolinum brattstromi and Lamellibrachia columna were used to history stages (Vogler and Monaghan, 2007). This approach has represent the outgroup taxa in phylogenetic analyses. The analyses proved immensely useful for identifying morphologically cryptic were conducted with the program MR.BAYES v. 3.1.2 (Ronquist and species in several poorly known deep-sea taxa (Vrijenhoek, 2009). Huelsenbeck, 2003). An appropriate substitution model for COI was We employ invertebrate primers that amplify DNA sequences determined using the MrModelTest2 procedure (Nylander, 2004) from mitochondrial cytochrome-c-oxidase subunit 1 (COI) within the program PAUP*v.4.02(Swofford, 2002). The site-specific (Folmer et al., 1994; Nelson and Fisher, 2000). This gene has general time reversible substitution model (GTR+SS) provided the already been used successfully in a number of studies to delineate best fit for the data obtained in this study. Bayesian analyses vestimentiferan (Chevaldonne´ et al., 2002; Feldman et al., 1998; utilized six chains, conducted for 5 Â 107 generations with a print Kojima et al., 2002) and Osedax species (Braby et al., 2007; and sampling frequency of 1000, and a burn-in of 2500. Each Fujikura et al., 2006; Jones et al., 2008). Here we use COI analysis was repeated five times and data were visualized using sequences to distinguish among 15 discrete evolutionary lineages TRACER v. 1.3 (Rambaut and Drummond, 2003)andAWTY of frenulates from the Gulf of Cadiz mud volcanoes. (Wilgenbusch et al., 2004) to determine the appropriate burn-in and ensure that the data had reached convergence. Trees were visualized with FIGTREE v. 1.2.2 (http://tree.bio.ed.ac.uk/software/ 2. Materials and methods figtree/). Saturation plots of nucleotide substitutions were created using the program DAMBE v. 5.0.25 (Xia and Xie, 2001). The plots 2.1. Sample collection and preservation (not shown) revealed significant saturation for transitions but not for transversions. For comparative purposes we included a specimen of Spiro- brachia cf. grandis that was provided and identified by Eve 2.4. Gauging MOTUs Southward. All other specimens were collected from 13 mud vol- canoes in the Gulf of Cadiz during several recent cruises